Equilibrium and kinetic binding analysis of the N-terminal domain of the Pf1 gene 5 protein and its interaction with single-stranded DNA.
The Pf1 gene 5 protein is a single-stranded DNA-binding protein that binds cooperatively to the viral strand of Pf1 DNA during replication. A variety of N-terminal fragments of the Pf1 gene 5 protein have been expressed and purified. We have identified an N-terminal single-stranded DNA-binding domain (residues 1 to 105) that is much more globular than the intact protein (1 to 144). Larger fragments (1 to 115) as well as smaller fragments (1 to 91) were unable to bind DNA effectively. Analysis of the truncated proteins by gel retardation and fluorescence anisotropy indicates that the N-terminal domain binds DNA with a reduced affinity, due principally to a reduction in cooperativity, and that binding is highly concentration-dependent. Kinetic analysis shows that the rates of association and dissociation of the N-terminal domain from the complex with DNA are faster than those observed for the intact protein. The results suggest that the flexible C-terminal domain of the Pf1 gene 5 protein plays an important role in protein-protein interactions that stabilise adjacent protein dimers in the DNA-protein complex.
Bogdarina
I
I
Biophysics Laboratories, University of Portsmouth, St Michaels, UK.
Fox
D G
DG
Kneale
G G
GG
D015374:Biosensing Techniques; D002352:Carrier Proteins / Q000235:genetics; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004586:Electrophoresis; D005454:Fluorescence Polarization; D017912:Inovirus / Q000235:genetics*; D007700:Kinetics; D058619:Maltose-Binding Proteins; D010446:Peptide Fragments / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D002352:Carrier Proteins; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D058619:Maltose-Binding Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C067863:gene 5 protein, Pseudomonas phage Pf1